Hepatic inducible nitric oxide synthase expression increases upon exposure to hypergravity

Abstract

Stimulation by a number of conditions, including infection, cytokines, mechanical injury, and hypoxia, can upregulate inducible nitric oxide synthase (iNOS) in hepatocytes. We observed that exposure to hypergravity significantly upregulated the transcription of the hepatic iNOS gene. The aim of this study was to confirm our preliminary data, and to further investigate the distribution of the iNOS protein in the livers of mice exposed to hypergravity. ICR mice were exposed to +3 Gz for 1 h. We investigated the time course of change in the iNOS expression. Hepatic iNOS mRNA expression progressively increased in centrifuged mice from 0 to 12 h, and then decreased rapidly by 18 h. iNOS mRNA levels in the livers of centrifuged mice was significantly higher at 3, 6, and 12 h than in uncentrifuged control mice. The pattern of iNOS protein expression paralleled that of the mRNA expression. At 0 and 1 h, weak cytoplasmic iNOS immunoreactivity was found in some hepatocytes surrounding terminal hepatic venules. It was noted that at 6 h there was an increase in the number of perivenular hepatocytes with moderate to strong cytoplasmic immunoreactivity. The number of iNOS-positive hepatocytes was maximally increased at 12 h. The majority of positively stained cells showed a strong intensity of iNOS expression. The expression levels of iNOS mRNA and protein were significantly increased in the livers of mice exposed to hypergravity. These results suggest that exposure to hypergravity significantly upregulates iNOS at both transcriptional and translational levels.

Figure 1. Effect of hypergravity exposure on inducible nitric oxide synthase (iNOS) mRNA expression in the livers of mice. Quantitative real-time RT-PCR analysis of iNOS mRNA was performed. Data are reported as the means±SE of 3 independent experiments in each group. The 3, 6, and 12 h groups displayed significantly higher iNOS mRNA levels in the liver than the control group (*P<0.01 and **P<0.001, Student t-test). In contrast, in the 18 and 24 h groups, hepatic iNOS mRNA was undetectable.

Figure 2. Effect of hypergravity exposure on inducible nitric oxide synthase (iNOS) protein expression in the livers of mice. A, The control group showed no cytoplasmic iNOS immunoreactivity in the hepatocytes, except for a demarcation of sinusoidal endothelial cells by iNOS. B, The 0 h group showed weak iNOS immunoreactivity in the cytoplasm of some hepatocytes (arrowheads) surrounding the terminal hepatic venules. C, In the 1 h group, the intensity and proportion of iNOS expression in the hepatocytes (arrowheads) were nearly identical to those of the 0 h group. D, The 3 h group revealed a significantly increased staining intensity of iNOS expression in the perivenular hepatocytes (arrows), compared with that of the 0 or 1 h groups. E, The 6 h group displayed an increased number of iNOS-positive hepatocytes, with a moderate to strong intensity of expression (double arrowheads). F, In the 12 h group, maximal expression of iNOS protein was observed and the number of iNOS-positive hepatocytes was increased. The majority of those cells exhibited a strong intensity (double arrowheads). G, The 18 h group showed no iNOS immunoreactivity in the hepatocytes. Polymer method. Original magnification, 150×.

Figure 3. Effect of hypergravity exposure on pro-inflammatory cytokine production in the livers of mice. ELISA was performed to measure the hepatic levels of cytokines. Data are reported as the means±SE of 3 independent experiments in each group. A, IL-1β. B, IL-6. C, IFN-γ. D, TNF-α. IL: interleukin; IFN-γ: interferon-γ; TNF-α: tumor necrosis factor-α. The centrifuged mice exhibited no significant changes in the concentrations of the 4 cytokines compared to those of the control mice (Student t-test).